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Preparation and Identification of TAT Modified Mouse and Human IFNγ

Author: HuDaQiang
Tutor: HeFengTian
School: Third Military Medical University
Course: Biochemistry and Molecular Biology
Keywords: Interferon gamma protein transduction domains prokaryotic expression protein purification immunohistochemistry blood-brain barrier ELISA
CLC: R392
Type: Master's thesis
Year: 2006
Downloads: 29
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Abstract

Interferon gamma(IFNγ) possesses the ability of anti-tumor and anti-virus,but the blood-brain barrier (BBB) can block it to enter brain,so the application of IFNγon treating brain tumors and virus infection is limited. Small region of the TAT protein from human immunodeficiency virus I which called protein transduction domains(PTD)is a kind of polypeptide,when fused with protein,it can effectively facilitate other molecules crossing many membrane structures including BBB,cell membrane, skin etc. As a small polypeptide, without cytotoxicity and immunogenicity,TAT PTD protein don’t affect the function of the fused proteins.So TAT modified IFNγmay be capable of passing through the BBB and applied to the treatment of brain tumor and virus infection. In this study,the cDNAs encoding TAT modified mouse and human IFNγ(mIFNγ-TAT and huIFNγ-TAT) were cloned and inserted into expression vector pQE-80L.The target proteins were expressed, purified and their functions on inhibiting cell proliferation were analyzed by MTT assay and BrdU-ELISA.The ability to tanslocate into cells for the IFNγ-TAT fusion proteins was identified by immunohistochemistry.The effects of mIFNγ-TAT crossing BBB was identified by detecting the concentration of mIFNγ-TAT in SD rat cerebrospinal fluid using ELISA.The major results and conclusions are summarized as follows:1. The cDNAs of mIFNγand huIFNγwere cloned and sequenced,which were consistent with the DNA sequences encoding mouse IFNγand human IFNγreported in GenBank.2. The cDNAs encoding mIFNγ-TAT and huIFNγ-TAT were amplified and cloned into pUC19 plasmid. DNA sequencing showed that the two fused cDNAs were accurate.3. After subcloned into expression vector pQE-80L and transformed into E.coli DH5α, the recombinant proteins mIFNγ,mIFNγ-TAT, huIFNγand huIFNγ-TAT were expressed in the present of IPTG,which were about 1.6×10~4,1.7×10~4,1.7×10~4 and 1.8×10~4 Daltons identified by SDS-PAGE and Western blot.The optimized parameters for mIFNγand

Preparation and Identification of TAT Modified Mouse and Human IFNγ

Abstract

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